Nakajima K , Tanizaki Y , Luu N , Zhang H , Shi YB .

ZIA HD001901-23 Intramural NIH HHS

             nucleosome assembly
             antibacterial humoral response

	Fig. 1. Identification of notochord-enriched, metamorphosis-induced genes (A) The Venn diagram representation of the RNA-Seq analyses. The mRNAs from stage-60-tail (St60), stage-63-tail (St63) and stage-63-notochord-removed-tail (St63dNC, i.e., the remaining tail after removing the notochord) are sequenced by RNA-Seq and compared among each other. The lined areas represent the expression of notochord-enriched genes. The shaded areas represent the expression of metamorphosis-induced genes. The lined and shaded area represents that of notochord-enriched, metamorphosis-induced genes. (B) The Venn diagram showing the result of the comparisons between St63 and St63dNC (solid line) and between St63 and St60 (dashed line) RNA-Seq data. Each sample was prepared from the mixture of 5 (St60), 5 (St63) and 8 (St63dNC) tails, respectively. The numbers of genes with a more than two-fold increase in St63, were indicated. (C, D) Expression profiles of the 630 notochord-enriched, metamorphosis-induced genes. Each gene is represented by a circle in the graph, as determined by the relative expression levels in the St63 (vertical axis) and St60 (C) or St63dNC (D). The names of the most highly expressed four genes in St63 (arrows) and expression levels of elongation factor 1 alpha 1 (EF1α; [ENSXETG00000001182], white arrowhead) are also showed in figure. The dashed line indicates equal expression level between two samples. mmp9-th; matrix metalloproteinase 9-th [ENSXETG00000033145], scppa2; secretory calcium-binding phosphoprotein acidic 2 [ENSXETG00000000337], mmp13; matrix metalloproteinase 13 [ENSXETG00000022190], olfm4; olfactomedin 4 [ENSXETG00000018715]. Data were the average of three technical replications. FPKM: fragments per kilobase of exon per million reads mapped.
	Fig. 2. The expression profiles of matrix metalloproteinases (MMPs). The expression levels of collagenases (A), gelatinases (B), stromelysins (C), membrane-type MMPs (D), and other MMPs (E) in stage-60-tail (St60), stage-63-tail (St63) and stage-63-notochord-removed-tail (St63dNC) were obtained from RNA-Seq data by using ENSEMBL database except for mmp7. The data for mmp7 was obtained from the same RNA-Seq data by using NCBI database because there is no accession number for mmp7 in ENSEMBL. The fold differences between St63 and St60 or St63dNC were indicated in figures. Samples were same as Fig. 1. Data were mean ± SD of three technical replications.
	Fig. 3. The expression profiles of olfm4 and scppa2 (A) The schematic representations of olfm4 gene structure with exons shown as boxes. (B) The expression levels of each exon of olfm4 from RNA-Seq as analyzed in Fig. 2. (C) The schematic representations of scppa2 gene structure with exons shown as boxes. (D) The expression levels of each exon of scppa2 from RNA-Seq as analyzed in Fig. 2. (E) The expression levels of olfm4 and scppa2 from RNA-Seq as analyzed in Fig. 2.(F) RT-PCR analysis of the expression levels of olfm4 and scppa2 in St60, St63, St63dNC and notochord (noto-A and noto-B) samples. The expression levels were shown as the copy number normalized against that of EF1α in each sample. The notochord samples were prepared from a mixture of 38 and 15 tadpoles, respectively, by using two different methods as described in previously (Nakajima et al. 2019). The fold changes between St63 and St60 or St63dNC (B and D-F), and between St63dNC and the average of the two notochord samples (F) were indicated. The samples of St60, St63 and St63dNC were the same as in Fig. 1. Data were mean ± SD of three technical replications.
	Fig. 4. Distinct localizations of olfm4 and scppa2 expression in the tail at the climax of metamorphosis. Cross sections of the tail at stage 62 were hybridized with sense olfm4 or scppa2 probes (A, A’, C, C’) or their antisense probes (B, B’, D, D’). Dark purple deposits indicate the sites of probe binding. Black pigments in some areas, e.g., spinal cord (SC), are melanin (see A, A’, C, C’). Note that olfm4 mRNA is expressed in the outer sheath cells (OS), but not in the connective tissue sheath (CT). Scppa2 mRNA is expressed in the OS and in the CT surrounding the notochord (NC). These same expression patterns were observed in three individual animals (not shown). Scale bars, 0.5 mm in A-D, 0.1 mm in A’-D’.
	Fig. 5. The up-regulated genes in the phagosome pathway. The metamorphosis-induced genes were showed in red and the genes induced specifically in notochord during metamorphosis were showed in purple. There were no genes which were expressed at 2 fold or higher level in the whole tail compared to the notochord-removed tail at stage 63 but not upregulated between stage 60 and 63, i.e., all notochord-enriched genes in this pathway were upregulated during metamorphosis. The genes whose expression was not changed were shown in light green.
	Fig. 6. Two potential models for notochord regression. MMP13 (13) expressed by outer sheath cells (solid green line) digests the notochordal sheath (solid red line). MMP9-TH (9TH) expressed by outer sheath cells digest the peri-notochordal basement membrane, thereby leading to the detachment of outer sheath cells from notochordal sheath, which in turn causes their apoptosis (dashed green lines). The ECM digestions by MMPs are indicated as yellow marks. Subsequently, two possible scenarios may occur. Scenario 1: The inner vacuolated cells are induced to undergo apoptosis (dashed gray circles) due to the loss of outer sheath cells. The notochord regression is mainly driven by the apoptosis of inner vacuolated cells. Scenario 2: Expression of olfm4 (olfm) as an extracellular matrix glycoprotein by the outer sheath cells facilitates the cell adhesion among inner vacuolated cells (purple arrow). The major driving force of notochord regression is the shrinkage of inner vacuolated cells.
	Caption in image.

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